Internal de novo initiation sites of the HCV NS5B polymerase and use thereof

ABSTRACT

To further define the nature of de novo initiation from the 3′-UTR, several distinct 3′-UTR&#39;s that harbor the conserved terminal 98 nucleotides, but have poly U/U-C tracts of different length were isolated and characterized. Reconstitution of de novo initiation by the mature NS5B with the different 3′-UTR RNA substrates revealed distinctively sized products that are consistent with internal initiation at specific sites within the polypyrimidine tract. These sites were mapped by demonstrating that nucleotide substitutions of the cytidylate residues in the poly U/U-C template affect the generation of specific products of the de novo initiation reaction. Moreover, initiation within the poly U/U-C template is also primed by GTP and an assay that evaluates inhibitors of this reaction as potential HCV therapeutics is claimed.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This claims priority to U.S. Provisional Application 60/198,793filed Apr. 21, 2000, the contents of which are fully incorporated byreference herein.

FIELD OF THE INVENTION

[0002] The present invention provides a de novo initiation sitecomprising a polypyrimidine tract having a cytidylate nucleotide or apoly-cytidylate (poly C) cluster located therein or adjacent thereto.This site provides a RNA template for assessing in vitro theRNA-dependent RNA polymerase (RdRp) activity of flavivirus.Particularly, the invention relates to de novo initiation sites of theNS5B protein of the hepatitis C virus and methods for identifyingspecific inhibitors thereof.

BACKGROUND OF THE INVENTION

[0003] Hepatitis C virus (HCV) is the major etiological agent ofpost-transfusion and community-acquired non-A non-B hepatitis worldwide.It is estimated that about 170 million people worldwide are infected bythe virus. A high percentage of carriers become chronically infected andmany progress to chronic liver disease, so called chronic hepatitis C.This group is in turn at high risk for serious liver disease such asliver cirrhosis, hepatocellular carcinoma and terminal liver diseaseleading to death.

[0004] HCV is an enveloped positive strand RNA virus in the Flaviviridaefamily. The single strand HCV RNA genome is 9600 nucleotides in lengthand has a single open reading frame (ORF) encoding a single largepolyprotein of about 3000 amino acids. In infected cells, thispolyprotein is cleaved at multiple sites by cellular and viral proteasesto produce structural and non-structural (NS) proteins. The structuralproteins (C, E1, E2 and E2-p7) comprise polypeptides that constitute thevirus particle (Hijikata et al., 1991; Grakoui et al., 1993(a)). Thenon-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) encode forenzymes or accessory factors that catalyze and regulate the replicationof the HCV RNA genome. Processing of the structural proteins iscatalyzed by host cell proteases (Hijikata et al., 1991). The generationof the mature non-structural proteins is catalyzed by two virallyencoded proteases. The first is the NS2-3 zinc-dependent metalloproteasewhich auto-catalyses the release of the NS3 protein from thepolyprotein. The released NS3 contains a serine protease domain at theN-terminal (Grakoui et al, 1993(b); Hijikata et al., 1993) and catalyzesthe remaining cleavages from the polyprotein. The released NS4A proteinhas at least two roles. First, forming a stable complex with NS3 proteinand assisting in the membrane localization of the NS3/NS4A complex (Kimet al., 1999) and second, acting as a cofactor for NS3 proteaseactivity. This membrane-associated complex, in turn catalyzes thecleavage of the remaining sites on the polyprotein, thus effecting therelease of NS4B, NS5A and NS5B (Bartenschlager et al., 1993; Grakoui etal., 1993(a); Hijikata et al., 1993; Love et al., 1996; reviewed inKwong et al., 1998). The C-terminal segment of the NS3 protein alsoharbors nucleoside triphosphatase and RNA helicase activity (Kim et al.,1995). NS5B is an RNA-dependent RNA polymerase (RdRp) that is involvedin the replication of HCV. It has been recognized that the NS3 proteaseand the NS5B polymerase activities constitute suitable enzymatic targetsto inhibit viral replication.

[0005] The non-coding RNA regions of the HCV genome are defined bydistinct 5′ and 3′ sequences (reviewed in Reed and Rice, 2000). The 5′extremity encodes an internal ribosome-entry site that folds into ahighly ordered secondary structure and directs cap-independenttranslation of the genomic RNA. The 3′ untranslated region is dividedinto three segments: (i) the highly conserved 3′-terminal 98 nucleotides(termed the X region) predicted to fold into a secondary structure ofthree stem-loop domains; (ii) a poly(U-U/C)-rich sequence of variablelength upstream of the X-region; and (iii) further upstream is a highlyvariable sequence 30-40 nt in length (Kolyakhov et al., 1996; Tanaka etal., 1996).

[0006] The initial step of viral RNA replication is recognition of the3′-end of RNA template by NS5B (RdRp), which may occur directly orindirectly with the help of cellular proteins (Lai, 1998; Strauss etal., 1999). HCV NS5B RdRp has an RNA-binding activity and preferentiallybinds poly(U) and poly(G) over poly(C) and poly(A) homopolymeric RNA(Yamashita et al., 1998).

[0007] It has recently been shown that NS5B can utilize the 3′-end98-nt, X region, of the HCV genome as a minimal authentic template (Ohet al., 2000). Furthermore, this RNA was used to characterize themechanism of RNA synthesis by the recombinant NS5B. The authors showthat NS5B forms a complex with the 3′-end of HCV RNA by binding to boththe poly(U-U/C)-rich and X regions of the 3′-untranslated region as wellas part of the NS5B-coding sequences. Within the X region, NS5B boundstem II and the single-stranded region connecting stem-loops I and II.Furthermore, NS5B initiated RNA synthesis from a specific site withinthe single-stranded loop I. They conclude that HCV NS5B initiates RNAsynthesis from a single-stranded region closest to the 3′-end of the Xregion, but do not disclose specific sequences that would induceinternal de novo initiation of the NS5B polymerase.

[0008] Sun et al., 2000 and WO 2000/33635 suggest that the NS5B alsocatalyses de novo RNA synthesis with a HCV RNA template. They furtherassert that “since the viral enzyme selectively added ATP as the firstnucleotide of the nascent RNA products, we conclude that HCV NS5Binitiated the RNA transcription by recognizing a uridylate present inthe HCV RNA fragment.” They go on to say that “it is possible that theenzyme recognized a uridylate present in the poly U/polypyrimidinetract.” However, Sun et al. fail to provide the exact sequence of the denova RNA polymerase initiation site present in the HCV RNA genome.

[0009] In contrast to the previous reports, Applicant has found that theHCV NS5B catalyses de novo initiation of the RNA template where theresulting nascent RNA products contained GTP as the first nucleotide. Inaddition, Applicant has data strongly suggesting that this de novoinitiation takes place internally along the RNA template, since theresulting nascent RNA products are smaller than the RNA template andcorrespond in length to RNA products having been initiated at distinctpositions corresponding to several cytidylate clusters positioned alongthe poly U tract of the HCV RNA template.

[0010] These results have implications for the mechanism of HCV RNAtranscription and the nature of HCV RNA templates in the infected cells.This site provides an RNA template for assessing in vitro the RdRpactivity of the NS5B protein and finding specific inhibitors thereof.

[0011] De novo initiation should be closer to the authentic mechanism ofRNA transcription during the disease state and in vivo infection. Theexact pattern of the de novo initiation site is disclosed herein and usethereof for design of an assay to measure NS5B RdRp activity. Theestablishment of such an assay will facilitate the analysis of theinitiation requirements and allow the testing of antiviral compoundsspecifically targeting de novo initiation of the HCV NS5B RNApolymerase.

SUMMARY OF THE INVENTION

[0012] The present invention provides the initiation site for de novo(primer-independent) RNA synthesis of an RNA-dependent RNA-polymerase,particularly for a flavivirus RdRp, more particularly for the HCV NS5Bpolymerase.

[0013] A first embodiment of the invention provides an initiation sitecomprising a polypyrimidine tract having at least one cytidylate residuelocated therein, or adjacent thereto.

[0014] A second embodiment of the invention provides a RNA template forprimer-independent RNA synthesis, this template comprising theinitiation site as described in the first embodiment and a further RNAportion suitable as a template for elongation of an initiationnucleotide along said template by said polymerase.

[0015] A third embodiment of the invention provides a method ofidentifying a compound that inhibits primer-independent de novo RNAsynthesis catalyzed by the HCV NS5B polymerase.

[0016] A fourth embodiment of this invention provides a method ofinhibiting primer-independent de novo RNA synthesis catalyzed by HCVNS5B polymerase where the compound is identified by the method asdescribed in the third embodiment of this invention.

[0017] A fifth embodiment of this invention provides a method ofinhibiting the replication of hepatitis C virus where the compound isidentified by the method as described in the third embodiment of thisinvention.

[0018] A sixth embodiment of this invention provides a method forproducing an anti-HCV compound whereby the compound is identifiedaccording to the method as described in the third embodiment of thisinvention.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0019]FIG. 1 is a schematic representation of the HCV 3′ ends used asRNA templates for the NS5B polymerase. HCV 3′ end cDNAs were cloneddownstream of a T7 RNA polymerase promoter and, following XbaI digestionand Mung Bean Nuclease treatment of DNA, RNA was synthesized in vitro asdescribed in Materials and Methods. For each RNA, the number at theright indicates the total length of the RNA while the numbers under thesequences indicate the nucleotide length of the different sections ofthe RNA. From 5′ to 3′ the RNAs contain a short leader of vectorsequence followed by the 3′-end of the NS5B coding region, the NS5B stopcodon and the HCV 3′ variable sequence; the polypyrimidine tract (thatvaries among the different RNA templates) and the highly conserved 3′-Xregion terminates the RNA templates.

[0020]FIG. 2A shows the major RNA products generated by HCV NS5B and the3′-UTR RNA produced that are shorter than the input template. Productsgenerated by NS5B polymerase with RNA templates: 24 (lane 2), 24-1 (lane3), 128-4 (lane 4), 130-21 (lane 5) or 150-41 (lane 6) were resolved adenaturing 8 M urea/5% polyacrylamide gel. RNA molecular weightstandards (M, lane 1) were used to interpolate the size of products. Thefilled circles represent the predicted location of radioactive productsthat correspond to full length RNA complementary to the template strand(i.e. de novo initiated at the 3′-end and run off at the 5′ end of thetemplate strand). The arrows highlight the major radioactive productsdetected.

[0021]FIG. 2B is a schematic representation of the distance from the Cstretches (2 Cs or more) present in the poly U/UC and the 5′ end oftemplates 24, 24-1 and 128-4; the distances correspond to the length ofthe major products from each of these templates and represent sites ofinternal de novo initiation by the HCV NS5B.

[0022]FIG. 3 is a schematic representation of the 3 mutated versions oftemplate 24-1 by site-directed substitution of the “CCCC” and/or the“CC” sequences from template 24-1 and the predicted effect on generationof HCV NS5B polymerization products. Non-substituted template 24-1 in astandard NS5B reaction produces 227 and 246 nt products. Template24-1(m1) has the 5′-proximal “CCCC” sequence mutated to “UUUU”. Intemplate 24-1(m2), the “CC” within the poly U/C is mutated to “UU”.Template 24-1(dm) encodes the double mutant with both m1 and m2modifications.

[0023]FIG. 4 shows a 8 M urea/5% acrylamide gel electrophoresis analysisof the reaction products using the mutants RNA templates as representedin FIG. 3. Nucleotide numbers shown on the right correspond to the twomajor products (246 and 227 nt) as described to originate from the 24-1RNA template. The size of the RNA markers (lane M) is indicated on theleft.

[0024]FIG. 5 shows the RNA products generated by NS5B polymerase in thepresence of [γ³²P]GTP or [γ³²p]ATP. De novo initiation reactionscontaining 25 nM (panels A, B and D) or 100 nM (panels C and E) of NS5Bpolymerase and 50 nM of RNA templates 24-1 (lanes 1), 24-1 (dm) (lanes2) or 128-4 (lanes 3) were used in polymerase reactions in the presenceof either [α³³P]UTP (panel A), [γ³²P]GTP (panels B and C) or [γ³²P]ATP(panels D and E). The products were analysed on a denaturing 8 M urea/5%acrylamide gel. Lane (M) displays radioactively labeled RNA molecularweight markers

[0025]FIG. 6 demonstrates the effect of GTP analog stimulation of NS5Bpolymerase in the de novo initiation reaction with the 24-1 HCV RNAtemplate. Various GTP analogs were added, at a final concentration of500 μM, to the NS5B polymerization reactions that contained: 100 μM(hatched bars) or 500 μM GTP (solid bars). Measurement of thestimulation by the analogs was normalized to the activity obtained withGTP alone. Reactions containing 600 μM or 1 mM GTP were also performed[labeled as the +500 μM GTP series]. Reactions were incubated for 2.5 hin the presence of 5 nM NS5B and 10 nM of RNA template 24-1. The effectof the various analogs on the reaction is represented by thefold-stimulation in activity relative to the activity obtained with 100μM or 500 μM GTP, respectively (these two are reference reaction bars).Note that guanosine, GpppG and 7-methyl GpppG all stimulate the de novoinitiation reaction. GTP 2′3′dialdehyde and ribavirin triphosphate areinhibitory. 5′-p-FSBA =5′-p-Fluorosulfonyl-benzoyladenosine.

[0026]FIG. 7 demonstrates the utility of one of the claimed RNAtemplates (24-1) in a HCV polymerase de novo initiation reaction thatmeasures the dose-dependent inhibition of compound I. Panel A: tablesummarizing the quantification of NS5B synthesized RNA from the 24-1template in the presence of indicated amounts of compound I, asdetermined through a DE-81 filter capture assay described in thematerials and methods. Panel B: plot of the %-inhibition versus compoundconcentration and the determination of the IC₅₀ value through non-linearregression analysis using the Hillequation:100−[(count_(inh)−count_(blank))/(count_(ctl)−count_(blank))×100].Panel C: RNA products resolved and visualized following electrophoresisin 8 M urea/5% acrylamide gels.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The following detailed description of the invention is providedto aid those skilled in the art practicing the present invention. Evenso, the following detailed description should not be construed to undulylimit the present invention as modifications and variations in theembodiments discussed herein can be made by those of ordinary skill inthe art without departing from the spirit or scope of the presentinventive discovery.

[0028] The contents of each of the references cited herein are hereinincorporated by reference in their entirety.

Definitions

[0029] Amino acid residues described herein are preferred to be in the“L”=0 isomeric form. However, residues in the “D” isomeric form may besubstituted for any L-amino acid residue, provided the desiredproperties of the polypeptide are retained.

[0030] All amino-acid residue sequences represented herein conform tothe conventional left-to-right amino-terminus to carboxy-terminusorientation.

[0031] All nucleotide sequence represented herein conform to theconventional left-to-right 5′ end to 3′ end orientation.

[0032] Conventional methods of gene isolation, molecular cloning, vectorconstruction, etc., are well known in the art and summarized, forexample, in Sambrook, J. et al., Molecular Cloning: A laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. One skilled in the art can readily reproduce theplasmid vectors described herein without undue experimentation employingthese methods. The various nucleic acid sequences, fragments, etc.,necessary for this and other purposes can be readily obtained ascomponents of commercially available plasmids, or are otherwise wellknown in the art and publicly available, or readily reproducible basedupon published information.

[0033] The phrase “consisting essentially of” when referring to aparticular nucleotide or amino acid means a sequence having theproperties of a given SEQ ID No. For example, when used in reference toa particular sequence, the phrase includes the sequence per se andmolecular modifications that would not affect the basic and novelcharacteristics of the sequence.

[0034] The term “cluster” as used herein defines a stretch of two ormore adjacent similar nucleotides aligned consecutively.

[0035] The terms “de novo” or “primer-independent” RNA synthesis areused interchangeably and refer to the ability of a polymerase to bind toa specific template, to prime RNA synthesis using a first initiatingnucleotide triphosphate (NTP) complementary to the initiation site, andelongate/extend the first nucleotide to transcribe the template withoutthe help of an extraneous oligonucleotide primer complementary to thetemplate.

[0036] A “derivative” of the HCV NS5B polypeptide or a fragment thereofmeans a polypeptide modified by varying the amino acid sequence of theprotein, e.g. by manipulation of the nucleic acid encoding the proteinor by altering the protein itself. Such derivatives of the natural aminoacid sequence may involve insertion, addition, deletion or substitutionof one or more amino acids, and may or may not alter the essentialactivity of the original HCV NS5B polypeptide. As mentioned above, theHCV NS5B polypeptide or protein of the invention includes any analogue,fragment, derivatives or mutant which is derived from a HCV NS5Bpolypeptide and which retains at least one property or othercharacteristic of the HCV NS5B polypeptide.

[0037] The terms “elongation” or “extension” are used interchangeablyand mean the consecutive addition of nucleotides as directed by acomplementary template of DNA or RNA that is carried out by anappropriate polymerase. In the particular context of this invention,elongation or extension is carried out on an RNA template by aflavivirus RNA-dependent RNA polymerase, particularly the HCV NS5B RdRp.

[0038] An “expression operon” refers to a nucleic acid segment that maypossess transcriptional and translational control sequences, such aspromoters, enhancers, translational start signals (e.g., ATC or AUGcodons), polyadenylation signals, terminators, and the like, and whichfacilitate the expression of a polypeptide coding sequence in a hostcell or organism.

[0039] The term “functional” as used herein implies that the nucleic oramino acid sequence is functional for the recited assay or purpose.

[0040] A “fragment” or “portion” of the HCV NS5B polypeptide means astretch of amino acid residues of sufficient length or an NS5Bpolypeptide having amino acids deleted therein, while retaining at leastone of its function such as binding to a template, priming, orelongation along a template.

[0041] The term “initiation site” means a site where the polymeraserecognizes a cytidine nucleotide or a nucleotide sequence comprising atleast one cytidine moiety. The initiation cytidylate nucleotidefunctions as a recognition site for primer-independent de novo RNAsynthesis on an RNA template catalyzed by HCV RNA-dependent RNApolymerase.

[0042] The term “initiation” refers the first step of RNA synthesis,that incorporates the initial 5′ position nucleotide of the nascent RNAchain. This reaction is also referred to as “priming”.

[0043] When applied to RNA, the term “isolated nucleic acid” refersprimarily to an RNA molecule encoded by an isolated DNA molecule asdefined above. Alternatively, the term may refer to an RNA molecule thathas been sufficiently separated from other nucleic acids with which itwould be associated in its natural state (i.e., in cells or tissues). Anisolated nucleic acid (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or host organism.

[0044] The terms “isolated protein” or “isolated and purified protein”refer primarily to a protein produced by expression of an isolatednucleic acid molecule of the invention. Alternatively, this term mayrefer to a protein that has been sufficiently separated from otherproteins with which it would naturally be associated, so as to exist in“substantially pure” form. “Isolated” is not meant to exclude artificialor synthetic mixtures with other compounds or materials, or the presenceof impurities that do not interfere with the fundamental activity, andthat may be present, for example, due to incomplete purification,addition of stabilizers, or compounding into, for example, immunogenicpreparations or pharmaceutically acceptable preparations.

[0045] “Nucleic acid” or a “nucleic acid molecule” as herein refers toany DNA or RNA molecule, either single or double stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form.

[0046] The term “NS5B” refers to a portion of the HCV genome locatednear the 3′ end of the viral genome that specifies the region encoding aprotein, termed the “NS5B protein”, “NS5B polypeptide”, “NS5Bpolymerase” or combinations of these terms which are usedinterchangeably herein. NS5B in its natural state, functions as anRNA-dependent RNA polymerase (RdRp). The nucleic acid region encodingthe NS5B protein may also be referred to as the “NS5B gene”. Thus, theterm “NS5B” may refer to either a nucleic acid molecule encoding theNS5B polypeptide, to an NS5B gene or to an NS5B polypeptide, or to anyportions thereof, depending on the context in which the term is used.NS5B may further refer to natural allelic variants, mutants andderivatives of either NS5B nucleic acid sequences or NS5B polypeptides.The NS5B nucleic acid, NS5B gene or NS5B protein referred to is afunctional polymerase, or to a non-functional polymerase that stillbinds to an appropriate template.

[0047] The term “oligonucleotide”, as used herein refers to primers andprobes of the present invention, and is defined as a nucleic acidmolecule comprised of two or more ribo- or deoxyribonucleotides,preferably more than three. The exact size of the oligonucleotide willdepend on various factors and on the particular application and use ofthe oligonucleotide.

[0048] The term “percent similarity”, “percent identity” and “percenthomology” when referring to a particular sequence are used as set forthin the University of Wisconsin GCG software program.

[0049] As used herein, the terms “polypyrimidine tract” and “poly U” areused interchangeably and refer to a stretch of consecutive pyrimidinenucleotides essentially consisting of uridylate residues. Preferably,the poly U stretch essentially consists of ≧70% uridylate residues. Morepreferably, the poly U tract essentially consists of ≧80% uridylateresidues. Most preferably, the poly U tract essentially consists of ≧90%uridylate residues.

[0050] As used herein the terms “poly U/C” and “poly U/U-C tract” areused interchangeably and refer to a poly U tract as defined aboveoptionally interrupted with at least one cytidylate residue, preferablytwo or more cytidylate residues and more preferably one or more clusterof cytidylate residues.

[0051] The term “plasmid” refers to an extrachromosomal genetic element.The starting plasmids herein are either commercially available, publiclyavailable on an unrestricted basis, or can be constructed from availableplasmids in accordance with published procedures. In addition,equivalent plasmids to those described are known in the art and will beapparent to the ordinarily skilled artisan.

[0052] The term “primer” as used herein refers to an oligonucleotide,either RNA or DNA, either single-stranded or double-stranded, eitherderived from a biological system, generated by restriction enzymedigestion, or produced synthetically which, when placed in the properenvironment, is able to functionally act as an initiator oftemplate-dependent nucleic acid synthesis. When presented with anappropriate nucleic acid template, suitable nucleoside triphosphateprecursors of nucleic acids, a polymerase enzyme, suitable cofactors andconditions such as a suitable temperature and pH, the primer may beelongated (extended) at its 3′ terminus by the addition of nucleotidesby the action of a polymerase or similar activity to yield a primerelongation (extension) product. The primer may vary in length dependingon the particular conditions and requirement of the application. Forexample, in diagnostic applications, the oligonucleotide primer istypically 15-25 or more nucleotides in length. The primer must be ofsufficient complementary to the desired template to prime the synthesisof the desired extension product, that is, to be able anneal with thedesired template strand in a manner sufficient to provide the 3′hydroxyl moiety of the primer in appropriate juxtaposition for similarenzyme. It is not required that the primer sequence represent an exactcomplement of the desired template. For example, a non-complementarynucleotide sequence may be attached to the 5′ end of an otherwisecomplementary primer. Alternatively, non-complementary bases may beinterspersed within the oligonucleotide primer sequence, provided thatthe primer sequences has sufficient complementarity with the sequence ofthe desired template strand to functionally provide a template-primercomplex for the synthesis of the extension product.

[0053] The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method.

[0054] “Recombinant DNA cloning vector” as used herein refers to anyautonomously replicating agent, including, but not limited to, plasmidsand phages, comprising a DNA molecule to which one or more additionalDNA segments can or have been added.

[0055] The terms “RNA synthesis” and “transcription” are usedinterchangeably and are defined by the specific steps taken by an RNApolymerase of: recognizing and binding to a template initiation site;priming by incorporating a first complementary nucleotide; and addingconsecutively complementary nucleotides to elongate the nascent RNAchain.

[0056] A “replicon” is any genetic element, for example, a plasmid,cosmid, bacmid, phage or virus, that is capable of replication largelyunder its own control. A replicon may be either RNA or DNA and may besingle or double stranded.

[0057] The term “specifically hybridize” refers to the associationbetween two single-stranded nucleic acid molecules of sufficientlycomplementary sequence to permit such hybridization under-pre-determinedconditions generally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention. To thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

[0058] The term “substantially pure” refers to a preparation comprisingat least 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

[0059] The term “tag”, “tag sequence” or “protein tag” refers to achemical moiety, either a nucleotide, oligonucleotide, polynucleotide oran amino acid, peptide or protein or other chemical, that when added toanother sequence, provides additional utility or confers usefulproperties, particularly in the detection or isolation, to thatsequence. Thus, for example, a homopolymer nucleic acid sequence or anucleic acid sequence complementary to a capture oligonucleotide may beadded to a primer or probe sequence to facilitate the subsequentisolation of an extension product or hybridized product. In the case ofprotein tags, histidine residues (e.g., 4 to 8 consecutive histidineresidues) may be added to either the amino- or carboxy-terminus of aprotein to facilitate protein isolation by chelating metalchromatography. Alternatively, amino acid sequences, peptides, proteinsor fusion partners representing epitopes or binding determinantsreactive with specific antibody molecules or other molecules (e.g., flagepitope, c-myc epitope, transmembrane epitope of the influenza A virushemaglutinin protein, protein A, cellulose binding domain, calmodulinbinding protein, maltose binding protein, chitin biding domain,glutathione S-transferase, and the like) may be added to proteins tofacilitate protein isolation by procedures such as affinity orimmunoaffinity chromatography. Chemical tag moieties include suchmolecules as biotin, which may be added to either nucleic acids orproteins and facilitates isolation or detection by interaction withavidin reagents, and the like. Numerous other tag moieties are known to,and can be envisioned by the trained artisan, and are contemplated to bewithin the scope of this definition.

[0060] The terms “transform”, transfect”, “transduce”, shall refer toany method or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, transfection,electroporation, micro-injection, PEG-fusion and the like.

[0061] The term “template” refers to an oligonucleotide of DNA, orpreferably RNA, that serves as one of the substrate for a polymerase.The sequence of a template is complementary to the sequence produced bythe polymerase during transcription.

[0062] Different “variants” of the HCV NS5B polypeptide exist in nature.These variants may be alleles characterized by differences in thenucleotide sequences of the gene coding for the protein, or may involvedifferent RNA processing or post-translational modifications. Theskilled person can produce variants having single or multiple amino acidsubstitutions, deletions, additions or replacements. These variants mayinclude inter alia: (a) variants in which one or more amino acidsresidues are substituted with conservative or non-conservative aminoacids, (b) variants in which one or more amino acids are added to theHCV NS5B polypeptide, (c) variants in which one or more amino acidsinclude a substituent group, and (d) variants in which the HCV NS5Bpolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the HCV NS5B polypeptide, such as, for example, anepitope for an antibody, a polyhistidine sequence, a biotin moiety andthe like. Other HCV NS5B polypeptides of the invention include variantsin which amino acid residues from one species are substituted for thecorresponding residue in another species, either at the conserved ornon-conserved positions. In another embodiment, amino acid residues atnon-conserved positions are substituted with conservative ornon-conservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques are known to the person having ordinary skillin the art. To the extent such allelic variations, analogues, fragments,derivatives, mutants, and modifications, including alternative nucleic.acid processing forms and alternative post-translational modificationforms result in derivatives of the HCV NS5B polypeptide that retain anyof the biological properties of the HCV NS5B polypeptide, they areincluded within the scope of this invention.

[0063] The term “vector” as used herein refers to a nucleic acidcompound used for introducing exogenous DNA into host cells. A vectorcomprises a nucleotide sequence which can encode one or more proteinmolecules. Plasmids, cosmids, viruses, and bacteriophages, in thenatural state or which have undergone recombinant engineering, areexamples of commonly used vectors, to which another genetic sequence orelement (either DNA or RNA) may be attached so as to bring about thereplication of the attached sequence or element.

Preferred Embodiments

[0064] According to the first embodiment of this invention, theinitiation site for de novo (primer-independent) RNA synthesis of theHCV NS5B RdRp preferably comprises a polypyrimidine tract having two ormore adjacent cytidylate residues located therein, or adjacent thereto.

[0065] Preferably, the RNA initiation site comprises a polypyrimidinetract having a cluster of cytidylate residues located therein, oradjacent thereto.

[0066] Preferably, the RNA initiation site comprises a polypyrimidinetract having one or more cluster of cytidylate residues located therein,or adjacent thereto.

[0067] More preferably, the RNA initiation site comprise a sequenceselected from the group consisting of: (P)_(n)(C)_(m); (C)_(m)(P)_(n);or (P)_(n)(C)_(m)(P)_(n), wherein P is a pyrimidine or an analogthereof, each n is independently 2 to 200 and m is 1 to 10.

[0068] Alternatively, the RNA initiation site preferably comprises a CCCor CCCC sequence adjacent to a polypyrimidine tract.

[0069] Still, most preferably, the RNA initiation site comprises asequence selected from the group consisting of:

[0070] U₁₃CU₃CCUUCU₃ (SEQ ID NO. 22);

[0071] U₁₁GU₂₉CU₃₀CCU₄CCU₁₀AUAUUCCUUCU (SEQ ID NO. 23);

[0072] U₁₄GGU₄₇CCU₅CCU₁₀AUAUUCCUUCU (SEQ ID NO. 24); and

[0073] U₁₃GU₅₈CCU₁₃AUAUUCCUUCU (SEQ ID NO. 25).

[0074] Preferably, the polypyrimidine tract is a poly(U) tractconsisting of equal to, or greater than 70% of uridylate residues, morepreferably 80%, most preferably 90%.

[0075] According to the second embodiment of the invention provides aRNA template for primer-independent RNA synthesis, this templatecomprising the initiation site as described in the first embodiment anda further RNA portion suitable as a template for said polymeraseelongation.

[0076] Preferably, the RNA template comprises a polypyrimidine tracthaving at least one cytidylate residue located therein, or adjacentthereto. More preferably, the initiation site of this template comprisestwo or more cytidylate residues, most preferably one or more cluster ofcytidylate residues.

[0077] Alternatively, the RNA template comprises a sequence of one ormore cytidylate residue adjacent to, the polypyrimidine tract,preferably two or more C residues, more preferably a CCC sequence andmost preferably a CCCC sequence upstream of the polypyrimidine tract.

[0078] According to the third embodiment of the invention, there isprovided a method of identifying a compound that inhibitsprimer-independent de novo RNA synthesis catalyzed by the HCV NS5Bpolymerase, comprising the steps of:

[0079] a) contacting a RNA template as described above, with the NS5Bpolymerase in the absence of a primer and in the absence of the compoundunder conditions permitting RNA synthesis, and determining the amount ofRNA thus formed;

[0080] b) contacting a RNA template as in a), with said NS5B polymerasein the absence of a primer and in the presence of the compound under thesame conditions as in a), and determining the amount of RNA thus formed;and

[0081] c) comparing the amount of RNA product formed in b) with that ofa);

[0082] wherein any reduction in the amount of RNA product formed in b)compared with that formed in a) indicates a compound that is aninhibitor of primer-independent de novo RNA synthesis catalyzed by theHCV NS5B polymerase.

[0083] Preferably, the compound inhibits binding of the NS5B polymeraseto the initiation site.

[0084] Alternatively, the compound inhibits priming of the NS5Bpolymerase once bound to the initiation site.

[0085] As a further alternative, the compound inhibits elongation by theRNA polymerase along the template.

[0086] According to the fourth embodiment of this invention, there isprovided a method of inhibiting primer-independent de novo RNA synthesiscatalyzed by HCV NS5B polymerase comprising the step of:

[0087] contacting the polymerase with a polymerase-inhibiting effectiveamount of a compound that inhibits primer-independent de novo RNAsynthesis catalyzed by the polymerase,

[0088] wherein the compound is identified by the method as described inthe third embodiment of this invention.

[0089] Preferably, the method provides inhibition of the binding of theNS5B polymerase to the initiation site.

[0090] Alternatively, the method provides inhibition of priming of theNS5B polymerase once bound to the initiation site.

[0091] As a further alternative, the method provides inhibition ofelongation by the RNA polymerase along the template.

[0092] According to the fifth embodiment of this invention provides amethod of inhibiting the replication of hepatitis C virus comprising thestep of:

[0093] contacting the hepatitis C virus with an antiviral effectiveamount of a compound that inhibits primer-independent de novo RNAsynthesis catalyzed by HCV NS5B polymerase,

[0094] where the compound is identified by the method as described inthe third embodiment of this invention.

[0095] Preferably, the method provides inhibition of the binding of theNS5B polymerase to the initiation site.

[0096] Alternatively, the method provides inhibition of priming of theNS5B polymerase once bound to the initiation site.

[0097] As a further alternative, the method provides inhibition ofelongation by the RNA polymerase along the template.

[0098] A sixth embodiment of this invention provides a method forproducing a anti-HCV compound comprising the step of:

[0099] identifying the compound according to the method as described inthe third embodiment of this invention.

EXAMPLES Materials and Methods Cloning of the HCV 3′ Sequences

[0100] The template 24 cDNA sequence (SEQ ID NO. 1), complementary tothe HCV plus strand 3′-end, was obtained by semi-nested RT-PCR performedon RNA extracted from the serum of an infected individual (HCV genotype1b). The sequence of the 3 oligonucleotides used in the RT-PCR were:

[0101] 5087: 5′-TCT AGA CAT GAT CTG CAG AGA GGC CAG TAT CAG CAC TCT C-3′(antisense), (SEQ ID NO.2), 6089: 5′-ATG GGG CAA AGG ACG TCC G-3′(external sense) (SEQ ID NO.3) and 8017: 5′-GGA CCA AGC TTA AAC TCA CTCCAA TCC-3′ (internal sense) (SEQ ID NO.4).

[0102] The final PCR product was then directly cloned into the pCR3vector (Invitrogen) downstream of the T7 RNA polymerase promoter. Fromthis cloned cDNA, a HindIII fragment was removed (essentially consistingof vector sequences present between the T7 promoter and the HCVsequences). The resulting DNA was used to synthesize template 24-1 (SEQID NO.5). The cDNA sequence encoding templates 128-4 (SEQ ID NO.6),130-21 (SEQ ID NO.7) and 150-41 (SEQ ID NO.8) were generated bycombining the 5′-portion (upstream of the poly U/U-C tract) of template24-1 cDNA with three different cDNA fragments obtained by semi-nestedRT-PCR performed on RNA extracted from an HCV infected liver (unknowngenotype). The oligonucleotides used for the amplifications were 5087(SEQ ID NO. 2), 8046: 5′-TCC ACA GTT ACT CTC CAG-3′ (external sense)(SEQ ID NO. 9) and 8038: 5′-TAG GCA TTT ACC TGC TCC CCA ACC-3′ (internalsense) (SEQ ID NO.10).

In Vitro Synthesis Of The RNA Templates

[0103] Plasmid DNA containing the 3′ HCV cDNA was linearized with XbaIand then treated with Mung bean nuclease to eliminate extraneousoverhanging DNA such that the run-off transcript synthesized by T7 RNApolymerase terminated with the authentic HCV 3′-end. RNA synthesis wasperformed, using the T7 RiboMAX large scale RNA production system(Promega), for 2-2.5 h at 37° C. followed by DNase digestion for 30 minat 37° C. After phenol-chloroform extraction and isopropanolprecipitation, the pelleted RNA was resuspended in DEPC-treated waterand passed through a Microspin G-50 column (Pharmacia). RNAconcentration was determined by OD260/280 measurement. Some of the RNAtemplates were size fractionated and purified by capillary gelelectrophoresis (Beckman Coulter P/ACE MDQ) on gels of 4 % (w/v)hydroxyethylcellulose, 20 mM TAPS, 7 M Urea, pH 6.3. Homogeneousfull-length RNA templates were used as substrates to confirm thegeneration of internal sites of de novo initiation by the NS5Bpolymerase.

Purification of Mature HCV NS5B Polymerase

[0104] The NS5B polymerase was produced as a hexa-histidine taggedprecursor in Sf-21 insect cells infected from a recombinant baculovirusconstruct (BacHTaA5B). This vector encodes the full-length HCV NS5B andan N-terminal hexa-histidine linked by a dodecapeptide motif thatconstitutes an NS5A/NS5B cleavage site. Processing of the precursorprotein with the heterodimeric NS3 protease/NS4A peptide-cofactor(Bartenschlager 1999) produces a mature form of the 591 amino acid NS5B(SEQ ID NO. 12). In summary, BacHTaA5B infected Sf-21 cell pellets wereresuspended in lysis buffer (25 mM Tris pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 2mM β-mercaptoethanol, 500 mM NaCl, 50% glycerol, 2%n-dodecyl-β-D-maltoside and a cocktail of protease inhibitors), Douncehomogenized, treated with DNase I, sonicated and then clarified bycentrifugation (105000 ×g, 45 min., 4° C.). The resulting supernatantwas diluted with 3 volumes of buffer A (25 mM Tris pH 7.5, 2 mMβ-mercaptoethanol, 10% glycerol,10 mM imidazole, 500 mM NaCl, 0.15%dodecyl-β-D-maltoside and a cocktail of protease inhibitors) and appliedto a Ni-NTA chelating resin (Qiagen). The HTaA5B protein was eluted by alinear (10-500 mM) imidazole gradient in buffer A, and then diluted withbuffer B (20 mM Tris pH 7.5, 20% glycerol, 2 mM β-mercaptoethanol, 1 mMEDTA, 0.15% n-dodecyl-β-D-maltoside) to reduce the NaCl concentration to300 mM. The HTaA5B was applied to a DEAE Sepharose column, to removenucleic acids and the flow-through was diluted two-fold with buffer B tofurther reduce the NaCl concentration to 150 mM for the subsequentHi-trap heparin chromatography. Purified HTaA5B was eluted with a200-1000 mM NaCl gradient from the Hi-trap heparin column.

[0105] The NS3/4A cleavage (Laplante et al., 2000) that generates themature NS5B uses a 1:50:1.25 molar ratio of NS3 protease: 4A cofactorpeptide: HTaA5B precursor in buffer B diluted with an equal volume ofbuffer C (50 mM NaPO₄ pH 7.8, 10% glycerol, 0.3 M NaCl, 0.1%n-dodecyl-β-D-maltoside). The reaction is performed at room temperaturefor 45 min. followed by a 5 hour incubation at 4° C. Following NS3/NS4Acatalyzed removal of the His-tag, the reaction mixture is supplementedwith 10 mM imidazole and batch-mixed with Ni-NTA resin to bind thecleaved His-tag tails and any uncleaved HTaA5B protein. The resin ispelleted by centrifugation and the supernatant (mature NS5B fraction) issubjected to Hi-trap heparin chromatography as described above toseparate the NS3 protease from NS5B RdRp. The NS5B fractionated byheparin chromatography is applied, in buffer B containing 800 mM NaCl,to a preparative Superose-12 gel filtration column to recover a highlypure NS5B.

Site-Directed Mutagenesis

[0106] Mutations within the poly (U/UC) were generated with theQuickChange Site-Directed Mutagenesis Kit (Stratagene) using the 24-1cDNA clone as template for PCR. The following complementary pairs ofoligonucleotides were used to generate:      (i) Mutant 24-1(ml): 5′-CCAATA GGC CAT TTT TTT TTT TTT TTT TTC TTT CCT TCT TTG GTG-3′ (SEQ ID NO.13) and 5′-GGA AAG AAA AAA AAA AAA AAA AAA TGG CCT ATT GGC CTG GAG-3′(SEQ ID NO. 14);      (ii) Mutant 24-1(m2): 5′-CCC CTT TTT TTT TTT TTCTTT TTT TCT TTG GTG GCT CCA TC-3′ (SEQ ID NO. 15) and 5′-GCC ACC AAA GAAAAA AAG AAA AAA AAA AAA AGG GGA TGG CC-3′ (SEQ ID NO. 16); and     (iii) Mutant 24-1(dm): 5′- CCA ATA GGC CAT TTT TTT TTT TTT TTT TTCTTT TTT TCT TTG GTG-3′ (SEQ ID NO. 17) and 5′-CAC CAA AGA AAA AAA GAAAAA AAA AAA AAA AAA ATG GCC TAT TGG-3′ (SEQ ID NO. 18).

HCV NS5B RNA Polymerization Reactions

[0107] Unless indicated otherwise, the standard reactions thatincorporated [α-³³P]UTP contained 5 nM of purified NS5B and 10-50 nM ofthe indicated RNA template (i.e.: 24, 24-1, 128-4, 130-21, 150-41 ormutant templates) incubated at 22° C. for 2.5 h in 22.5 mM Tris-HCl pH7.5, 5 mM MgCl₂, 1 mM EDTA, 1.5 mM DTT, 30 mM NaCl, 0.33%N-Dodecyl-β-D-Maltoside, 0.01% Igepal CA-630, 5% DMSO, 5 μCi [α-³³P]UTP,10 μM UTP, 500 μM of the three other rNTPs and 0.16 U/μl RNase inhibitor(Promega). NS5B polymerase reactions that incorporated γ-³²P into theradioactively labeled 5′product were supplemented with either 8 μCi of[γ-³²P]GTP or [γ-³²P]ATP in place of [α-³³P]UTP in the standardreaction. The reactions were then supplemented with PK buffer (50 mMTris HCl pH 7.5 150 mM NaCl, 0.5% SDS) and treated with Proteinase K(0.8 μg /μl) and glycogen (0.1 μg/μl) for 30 min at 37° C. RNA wasextracted with phenol-chloroform and precipitated with isopropanol. TheRNA pellet was dissolved in 10-15 μl of denaturing solution (80%formamide, 10 mM EDTA), heated for 10 min at 70-75° C. and analysed ondenaturing 8 M urea/5% polyacrylamide gels. Gels were dried andradioactive products visualized with a phospho-imager (MolecularDynamics).

Testing of GTP Analogs And Other Inhibitors In The De Novo InitiationReaction

[0108] m7GpppG and GpppG were purchased from Ambion; Ribavirinmonophosphate and Ribavirin triphosphate were purchased from Chemgenes(Ashland, Mass.); ddGTP was from Amersham; Caged GTP was from MolecularProbes. All other GTP analogs were from Sigma/Aldrich. Each of theanalogs was added (at 500 μM) to the NS5B polymerization reactioncontaining either: 100 μM or 500 μM GTP (reference reactions).Stimulation by GTP itself, as a positive control, was performed suchthat the reactions then contained 600 μM or 1 mM GTP (respectivelylabeled as the +500 μM GTP bars). The standard polymerase reactionsincorporating [α-³³P]UTP were incubated for 2.5 h in the presence of 5nM NS5B and 10 nM of RNA template 24-1. The reactions were stopped byspotting 8 μl on a DE81 filters which were then washed extensively (3×)with 1M NaPO₄ pH 7 and dried. The radioactivity incorporated intoproduct RNA that was retained on the DE-81 filters was measured in ascintillation counter. The effect of the various analogs on the reactionis represented by the fold stimulation of activity compared to 100μM or500μM GTP (reference reaction bars).5′-p-FSBA=5′-p-fluorosulfonyl-benzoyladenosine

Example 1 Template Motifs in the Poly U/C Segment Correspond to Sites ofDe Novo Initiation

[0109] Primer-independent, de novo initiation of RNA synthesis by theHCV NS5B polymerase occurs on templates of various origin (Oh et al.,1999; Oh et al., 2000; Zhong et al., 2000; Luo et al., 2000; and WO2000/33635). We have confirmed this observation both with templates thatmimic the 3′-end of the plus-strand genome and the 3′-end of theminus-strand anti-genome. In contrast to the products that are generatedby NS5B with non-specific RNA templates (Luo et al., 2000) or RNAtemplates that mimic the 3′-end of the minus-strand, the predominantproducts from templates that correspond to the 3′-end of the plus-strandare significantly shorter than the unit length template. FIG. 1schematically displays the various RNA substrates with highlightedregions of the 3′-UTR, that were used in this study. The onlydistinction between templates 24 and 24-1 is the length of RNA upstreamof the poly-pyrimidine (poly U/U-C) tract. The remainder of the RNAtemplates (128-4, 130-21 and 150-41) are slight variations of the 24-1sequence that contain poly U/U-C tracts of various length that wereamplified and cloned from an HCV infected liver. HCV NS5B polymerasereactions that used the 415 nt RNA template 24, consistently producedtwo major radioactive products of approximately 292 and 311 nt (FIG. 2A,lane 2 denoted with arrows). A 415 nt product that corresponds to arun-off transcript initiating at the terminal nucleotide of the3′-template was produced (FIG. 2A, lane 2 denoted by filled circle), butto a significantly lesser extent.

[0110] The origin of the RNA products that are shorter than the inputtemplate was investigated through the use of slightly modified RNAtemplates. Interestingly, the sub-template size products were specificto reactions that contained templates mimicking the 3′-end of the HCVplus-strand. In reactions that used the 3′-end of the HCV minus-strandas a template (341 nts), none of the major products were shorter thanthe input template (data not shown; Oh et al., 1999). The possibleorigin of non-degraded products that were shorter than input templatemay be from: (i) premature termination of RNA polymerization thatinitiated de novo at the 3′ terminal nucleotide of the template; or (ii)run-off transcription that initiated at sites remote (upstream) from the3′-end of the template. In order to distinguish between these twopossibilities, we slightly modified template 24. An alteration at the5′-end constituted template 24-1 and reduced the length of RNA upstreamof the poly U/U-C tract from 292 to 227 nucleotides, −a 65 ntdifference. The two major products generated by HCV polymerase with thetruncated 24-1 template were approximately 227 and 246 nt long (FIG. 2A,lane 3 highlighted by two arrows). These products were also shorter thanpredicted from the complete transcription of a 350 nt unit lengthtemplate (FIG. 2A, lane 3 filled circle), and consistent with theresults obtained with template 24. Moreover, the difference in lengthbetween the pair of products generated from template 24 and 24-1 wasapproximately 65 nt. This differential coincides with the length of thealteration that distinguishes the 5′-end of template 24 from template24-1 and suggests that the pair of major products resulted from HCVpolymerase directed transcription that initiated at internal sites andran-off the different 5′-ends. We were careful to ensure that thetemplate preparation contained full length RNA template (and was notcontaminated with a small amount of RNA that may have prematurelyterminated during substrate preparation by the T7 RNA transcription) byemploying high resolution capillary gel electrophoresis to purify fulllength template RNA substrate.

Example 2 Position Of The Poly U/C Initiation Sites From The 5′-EndPredicts The Length Of The Synthesized Run-Off RNA Product

[0111] In a further effort to localize the potential initiation siteswithin the HCV 3′ plus-strand UTR, we modified the 24-1 RNA to generatetemplates 128-4, 130-21, and 150-41. The three templates and 24-1 sharethe similar sequence upstream and downstream of the poly-pyrimidinetract, but have the poly U/U-C portion increased from 25 to 101, 93, and98 nucleotides, respectively (FIG. 1). In polymerization reactions usingthe mature HCV NS5B, the major products generated from each of thesetemplates were shorter than input template RNA (FIG. 2A, lanes 4-6,compare position of arrows with location of filled circles). At 425-nt,template 128-4 was the longest model substrate used in these studies.Four major products (with estimated nucleotide lengths: 226, a 300/306doublet, and 323) were generated by HCV NS5B from the 128-4 RNA.Assuming a run-off transcript that terminates with a sequencecomplementary to the 5′ end of the template, the 226, 300, 306, and 323products are all predicted to initiate at template cytidylate doubletswithin the 128-4 template polypyrimidine tract (FIG. 2B). The 226 ntproduct initiates at the stretch of cytidylates 5′ to the poly U/U-Ctract, and migrates slightly quicker than the 227 nt product generatedby template 24-1 (which encodes a 4 cytidylate stretch rather than the 3C stretch encoded by the three other templates). The intense bandmigrating in the range of 300-310 nt range corresponds to a predicteddoublet originating from the two pairs of cytidylates in the middle ofthe poly U/C tract; and the 323 nt product represents RNA initiated atthe 3′ CC motif in the poly U/C tract.

Example 3 mutational analysis

[0112] In order to establish a role for the template ‘CC’ motifs aspotential sites of initiation on the HCV 3′ UTR, the two CC motifs oftemplate 24-1 were replaced with U residues. FIG. 3 depicts the threemutant templates used in this example. Template 24-1 (m1) (SEQ ID NO.18) harbors a four nucleotide substitution at the 5′ portion of the polyU/C tract that was predicted to abolish initiation and generation of the227 nt product. Template 24-1 (m2) (SEQ ID NO. 19) has a two nucleotidesubstitution of the CC motif in the poly U/C tract that was predicted toeliminate the 246 nt product, and RNA template 24-1(dm) (SEQ ID NO. 20)harbors both the (m1) and (m2) mutations. FIG. 4 displays the productsof primer-independent RNA synthesis by the HCV NS5B with each of thesetemplates. Relative to unmodified 24-1, the 24-1(m1) template does notgenerate the 227 nt product. The 24-1(m2) template substantially reducedthe production of 246 nt RNA, and the 24-1 (dm) double mutantsignificantly decreased the production of both 246 and 227 nt species.Minor, less intense products of internal initiation are still visibleand represent products that were initiated at alternative cytidylateswithin the template.

Example 4 A Role For GTP in Primer-Independent RNA Synthesis By HCV NS5B

[0113] The characteristic products of NS5B primer-independent RNAsynthesis from HCV 3′UTR templates are easily detected using [α-³³P]UTPprecursors that incorporate multiple ³³P isotopes into the synthesizedRNA backbone. Earlier studies have shown that the priming nucleotideincorporated into the RNA product by HCV NS5B maintains the 5′tri-phosphate by the use of [γ-P³²]NTPs (Zhong et al., 2000). In orderto further substantiate the role of template “CC” motifs as sites of denovo initiation by the HCV NS5B, we reconstituted the in vitro NS5Bpolymerase reaction with either [γ-P³²]GTP or [γ-P³²]ATP as the onlyradiolabeled tracer in the reaction. FIG. 5A displays the characteristicproducts (described above) that resulted from [α-P³³]UTP incorporationby 25 nM of NS5B polymerase with the 24-1 (lane 1), 24-1dm (lane 2), and128-4 (lane 3) templates. FIG. 5B shows that the use [γ-P³²]GTP in thereaction as the radiolabel tracer, rather than [α-³³P]UTP, dramaticallydecreased the intensity of the products, supporting the proposal thatonly one mole of gamma-phosphate was incorporated per mole of RNAproduced . Notwithstanding the decrease in product intensity, theproducts characteristic of initiation at template poly U/C “CC” motifswere detected with the [γ-P³²]GTP (FIG. 5 B) and were clearly evidentfrom reactions catalyzed by higher (100 nM) concentrations of NS5B (FIG.5C). The specificity of HCV NS5B in utilizing GTP as a primer attemplate “CC” motifs is highlighted by the lack of products detectedwith reactions that were reconstituted with [γ-P³²]ATP as the soleradioactive tracer (FIG. 5D and E).

Example 5 Evaluating the Effect of GTP Analogs on Primer-Independent RNASynthesis by HCV NS5B

[0114] Primer-independent RNA synthesis by HCV NS5B is stimulated byhigh (0.5 to 1 mM) concentrations of either GTP or ATP (Lohmann et al.,1999; Oh et al., 1999; Luo et al., 2000; Zhong et al., 2000). The NS5Band HCV 3′-UTR templates described in this work, showed a similaractivation by high concentrations of GTP. FIG. 6 depicts the results oftwo basic NS5B polymerase reactions reconstituted with the 24-1 templateRNA. The hatched bars represent reactions primed with a basal level of100 μM GTP in the reaction, and the closed bars represent reactionsprimed with a basal level of 500 μM GTP. Though the 100 μM GTP reactionproduces less product, the fold-stimulation in product intensity (asmeasured by the incorporation of [α-³³P]UTP) for each of the tworeference reactions is normalized to 1. Hence, the fold stimulationprovided by a supplement of 500 μM GTP is greater in the reaction with100 μM basal GTP, than in the reaction with 500 μM GTP, and reflectspreviously reported Km values for GTP in primer-independent RNAsynthesis (Lohmann et al., 1999; Luo et al, 2000; Zhong et al., 2000).

[0115] A number of analogs were examined as potential substitutes forthe stimulating GTP in the de novo initiation reaction with NS5B and the24-1 RNA template. The di-nucleotide cap analogs GpppG and m7 GpppG werethe most efficient analogs in stimulating de novo initiation, followedby guanosine, caged-GTP (fluorescently-tagged y-phosphate GTP), GMP andGDP-γ-S. The reactive GTP 2′3′-dialdehyde was a clear inhibitor.Moreover, the apparent consequence of supplementing 500 μM ribavirintri-phosphate in the reference reactions was a moderate level ofinhibition.

Example 6 Use of the HCV 3′UTR RNA Template 24-1 in A NS5B De NovoInitiation Reaction to Evaluate Inhibitors as Potential Anti-HCVTherapeutics

[0116] The assay presented in Example 1 was carried out for the purposeof screening potential inhibitors of the HCV NS5B polymerase and theireffect on de novo initiation from the specific sites (described above)on the HCV 3′UTR RNA template 24-1. Compound I was added, at theindicated concentrations (FIG. 7), to the standard HCV polymerasereaction with template 24-1 as detailed in the materials and methods.For quantification purposes, 8μL of the reaction were spotted on a DE81filter paper. After 3 washes (10 min each) in Na₂HPO₄/NaH₂PO₄ 1M pH 7,the filter was rinsed in water, then in EtOH and left to dry. Boundradioactivity, which is a measure of the amount of RNA produced in theprimer-independent NS5B RNA polymerase reaction, was quantified byliquid scintillation counting (FIG. 7A).

[0117] The % inhibition was calculated with the following equation:

100−[(count_(inh)-count_(blank))/(count_(ctl)-count_(blank))×100].

[0118] A non-linear curve fit with the Hill model was applied to theinhibition-concentration data, and the 50% effective concentration(IC₅₀) was calculated by the use of SAS software (Statistical SoftwareSystem; SAS Institute, Inc. Cary, N.C.).

[0119] The % inhibition is plotted versus the compound concentration toobtain an IC₅₀ of 5.5 μM for compound I. Moreover, the specificinhibition of NS5B in the production of the 227 and 246 nt species(products stemming from internal de novo initiation) from template 24-1demonstrate the utility of these compounds in evaluating specificinhibitors of HCV RNA transcription.

Discussion

[0120] The examples of the present invention provide template-strandinitiation sites comprising a polypyrimidine tract that are specificallyrecognized by RNA-dependent RNA polymerases, particularly the HCV NS5Bpolymerase. The precise initiation sites on the template strands areidentified as cytidylate or poly-cytidylate clusters located in oradjacent to a polyuridylate tract. To date, there has been no explicitfunction associated with the poly U/U-C tract or a characterization forits role in the interaction with other HCV encoded activities. Thoughthe poly U/U-C tract is a highly conserved segment of the HCV genome(Choo et al., 1989), the length of this segment varies, as exemplifiedby the cloning of different sized segments from the RNA genomes of anHCV-infected liver (Example 1). A feature of all these poly U/U-C tractsis the presence of short C or CC motifs that are located both at the5′-end and within the long poly U stretches. This sequence organizationis characteristic of all the genomic HCV RNAs examined. The examples ofthe present invention provide a specific role for the poly U/U-C tractand highlight the preference of the HCV NS5B polymerase for de novoinitiation specifically at the template C motifs. This is the firstevidence of a functional role for the 3′UTR poly U/U-C tract in HCVRNA-dependent RNA transcription and clearly demonstrates the requirementfor the C motifs, thereby providing an explanation for the conservationof this distinctive organization among HCV RNA genomes.

[0121] Primer-independent, de novo initiation of RNA synthesis is a wellcharacterized reaction of the HCV NS5B polymerase (Oh et al., 1999;Zhong et al, 2000; Luo et al., 2000; Sun et al., 2000), which isconserved among related flaviviral NS5B polymerases (Kao et al., 1999).The precise sites for de novo initiation are determined by thecomposition of the template sequence. NS5B RNA substrates that mimic the3′-end of the HCV RNA negative strand, and terminate with a 3′-C, serveas templates to generate products that contain 5′ GTP as the initiatingnucleotide on the product strand (Oh et al., 1999; Luo et al, 2000). Incontrast, HCV NS5B RNA templates comprising the 3′-end of the HCVplus-strand generate different products. Oh et al. 2000 and Sun et al.2000 demonstrate that RNA templates containing the poly U/U-C tract andthe 3′-UTR region, are transcribed by the HCV NS5B into RNA productsthat are smaller than the unit length template. Oh et al., 2000 suggestthat these products initiate at a site remote from the template 3′-endand prematurely terminate. Sun et al. 2000 and WO 2000/33635, publishedon 15 June 2000, disclose and claim initiation sequences specific fortemplate-independent RNA synthesis catalyzed by HCV polymerase. Theyclaim a distinct sequence in the HCV 3′-UTR template as a specificrecognition site for de novo initiation by the HCV NS5B polymerase.Despite their observation that non-specific templates comprising acluster of cytidylates also serve as substrate templates for HCV NS5B denovo initiation, the data presented explicitly argues that the HCVspecific RNAs utilize a template uridylate as the initiation site. Someexperimental data suggest that the polymerase has greater affinity forpoly C than poly U. However, in the case of their experiments with HCVRNA templates, this data is contradicted by the complete lack ofincorporation of [γ³²P]-GTP in resulting RNA products. They concludethat “the resulting nascent RNA products, smaller than the templateused, contained ATP as the first nucleotide suggesting that atranscription initiation site in the HCV RNA genome is uridylate”. Theyexplain the phenomenon of smaller RNA products on the basis of variousstages in RNA synthesis which leads the reader to believe that theinitiation observed is not carried out internally at different sites ofthe molecule but takes place at a single site:

[0122] “the major products of the reactions were about 370 nt in lengthi.e. about 100 bases smaller than the template. Meanwhile several minorRNA products, smaller than the template and the major RNA product, werepresent in the reaction, indicating various stages in RNA synthesis”. ..

[0123] They do not remotely suggest the possibility that the initiationsites may be scattered along the RNA, leading to RNA products ofdifferent length corresponding to the position of each initiation site.It is clear from the contradictory data, and lack of explanationthereof, that Sun et al. do not disclose the inventive concept of thepresent invention. Sun et al. were not in possession of the significanceof the C cluster with a poly U tract as the specific site for de novoinitiation of HCV NS5B polymerase. In addition, details lackingconcerning the types of templates used (such as the specific sequence oftheir poly U/C template) provides no information whether their templatecontained a cluster of C within a poly U stretch or simply a stretch ofU followed by a stretch of C. Even then, the data suggest that in a HCVtemplate, U is the site of initiation and not C.

[0124] The examples of this invention unambiguously identify the Cmotifs of the HCV 3′-UTR poly U/UC tract as template directed initiationsites. The different lengths of the major run-off transcription productsgenerated by the HCV NS5B from the templates labeled: 24, 128-4, 130-21and 150-41 in Example 1, precisely correlate with the position of the Cmotifs in the poly U/U-C tract in the respective templates. Moreover,the subtle substitution of uridylates for specific cytidylates withinone of the templates in Example 3, abolished the major products that arepredicted to originate from the corresponding C motifs. Finally, thedemonstration in Example 4 that a GTP constitutes the 5′-initiatingnucleotide on the product RNA, as supported by the incorporation of a[γ³²P]-GTP, highlights a specific role for the poly U/U-C templatecytidylate residues, in contrast to other claims (Sun et al., 2000. andWO 2000/33635; Zhong et al, 2000)

[0125] The distinction and utility of the present invention are bothapparent in the subsequent evaluation of various compounds as agonistsor antagonists of the GTP stimulated HCV NS5B de novo initiationreaction. Example 5 highlights compounds that are analogs of GTP,capable of either stimulating (such as the GpppG or 7-methyl GpppG) orinhibiting (such as GTP 2′3′-dialdehyde or ribavirin triphosphate) theNS5B reaction using these templates. Moreover, the sensitivity of thereaction to more potent inhibitors is evident from Example 6 wherein theNS5B catalyzed generation of the characteristic product using the HCVRNA template is specifically inhibited by a compound with an IC₅₀ in thelow micromolar range. The in vitro reconstituted de novo initiationreaction is closer to the authentic mechanism of HCV RNA transcriptionand replication in vivo. The further discovery of de novo initiationsites and use in the establishment of an assay to measure a specificstep in the NS5B RNA-dependent RNA polymerase reaction, disclosedherein, permitted the analysis of the initiation requirements. Theutility of the RNA templates and sites that we have described are asignificant part of the growing arsenal of tools used in the search forantiviral compounds specifically targeting the initial step of RNAsynthesis by the HCV NS5B RNA polymerase.

1 25 1 415 DNA Artificial Sequence misc_feature cDNA 1 gggagacccaagcttggtac cgagctcgga tccactagta acggccgcca gtgtgctgga 60 attcggcttggaccaagctt aaactcactc caatcccggc tgcgtcccgg ctggacttgt 120 ccggctggttcgttgctgga tacaacgggg gagacatata tcacagcctg tctcgtgccc 180 gaccccgttggtttatgttg tgcctactcc tactttctgt aggggtaggc atttacctgc 240 tccccaaccgatgaacgggg ggctaaacac tccaggccaa taggccatcc cctttttttt 300 tttttctttccttctttggt ggctccatct tagccctagt cacggctagc tgtgaaaggt 360 ccgtgagccgcatgactgca gagagtgctg atactggcct ctctgcagat catgt 415 2 40 DNAArtificial Sequence misc_feature oligonucleotide 2 tctagacatg atctgcagagaggccagtat cagcactctc 40 3 19 DNA Artificial Sequence misc_featureoligonucleotide 3 atggggcaaa ggacgtccg 19 4 27 DNA Artificial Sequencemisc_feature oligonucleotide 4 ggaccaagct taaactcact ccaatcc 27 5 350DNA Artificial Sequence misc_feature template 24-1 5 gggagacccaagcttaaact cactccaatc ccggctgcgt cccggctgga cttgtccggc 60 tggttcgttgctggatacaa cgggggagac atatatcaca gcctgtctcg tgcccgaccc 120 cgttggtttatgttgtgcct actcctactt tctgtagggg taggcattta cctgctcccc 180 aaccgatgaacggggggcta aacactccag gccaataggc catccccttt tttttttttt 240 ctttccttctttggtggctc catcttagcc ctagtcacgg ctagctgtga aaggtccgtg 300 agccgcatgactgcagagag tgctgatact ggcctctctg cagatcatgt 350 6 425 DNA ArtificialSequence misc_feature template 128-4 6 gggagaccca agcttaaact cactccaatcccggctgcgt cccggctgga cttgtccggc 60 tggttcgttg ctggatacaa cgggggagacatatatcaca gcctgtctcg tgcccgaccc 120 cgttggttta tgttgtgcct actcctactttctgtagggg taggcattta cctgctcccc 180 aaccgataaa cggggagcca aacactccaggccaataggc catccctttt tttttttgtt 240 tttttttttt tttttttttt tttttttctttttttttttt tttttttttt ttttttttcc 300 ttttcctttt ttttttatat tccttctggtggctccatct tagccctagt cacggctagc 360 tgtgaaaggt ccgtgagccg catgactgcagagagtgctg atactggcct ctctgcagat 420 catgt 425 7 417 DNA ArtificialSequence misc_feature template 130-21 7 gggagaccca agcttaaact cactccaatcccggctgcgt cccggctgga cttgtccggc 60 tggttcgttg ctggatacaa cgggggagacatatatcaca gcctgtctcg tgcccgaccc 120 cgttggttta tgttgtgcct actcctactttctgtagggg taggcattta cctgctcccc 180 aaccgataaa cggggagcca aacactccaggccaataggc catccctttt tttttttttt 240 ggtttttttt tttttttttt tttttttttttttttttttt tttttttttc ctttttcctt 300 ttttttttat attccttctg gtggctccatcttagcccta gtcacggcta gctgtgaaag 360 gtccgtgagc cgcatgactg cagagagtgctgatactggc ctctctgcag atcatgt 417 8 422 DNA Artificial Sequencemisc_feature template 150-41 8 gggagaccca agcttaaact cactccaatcccggctgcgt cccggctgga cttgtccggc 60 tggttcgttg ctggatacaa cgggggagacatatatcaca gcctgtctcg tgcccgaccc 120 cgttggttta tgttgtgcct actcctactttctgtagggg taggcattta cctgctcccc 180 aaccgataaa cggggagcca aacactccaggccaataggc catccctttt tttttttttg 240 tttttttttt tttttttttt tttttttttttttttttttt tttttttttt ttttttttcc 300 tttttttttt tttatattcc ttctggtggctccatcttag ccctagtcac ggctagctgt 360 gaaaggtccg tgagccgcat gactgcagagagtgctgata ctggcctctc tgcagatcat 420 gt 422 9 18 DNA Artificial Sequencemisc_feature oligonucleotide 9 tccacagtta ctctccag 18 10 24 DNAArtificial Sequence misc_feature oligonucleotide 10 taggcatttacctgctcccc aacc 24 11 1884 DNA Artificial Sequence misc_feature HTaA5Bpolymerase 11 atg tcg tac tac cat cac cat cac cat cac gat tac gat atccca acg 48 Met Ser Tyr Tyr His His His His His His Asp Tyr Asp Ile ProThr 1 5 10 15 acc gaa aac ctg tat ttt cag ggc gcc atg gat ccg gaa ttcgag gac 96 Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe GluAsp 20 25 30 gtc gtc tgc tgc tcg atg tcc tac aca tgg aca ggc gcc ctg atcacg 144 Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr35 40 45 cca tgc gcc gcg gag gaa agc cag ctg ccc atc aac gcg ttg agc aac192 Pro Cys Ala Ala Glu Glu Ser Gln Leu Pro Ile Asn Ala Leu Ser Asn 5055 60 tct ttg gtg cgt cat cgc aac atg gtc tat tcc aca aca tcc cgc agc240 Ser Leu Val Arg His Arg Asn Met Val Tyr Ser Thr Thr Ser Arg Ser 6570 75 80 gcg gcc ctg cgg cag aag aag gtc acc ttt gac aga ctg caa gtc ctg288 Ala Ala Leu Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu 8590 95 gac gac cac tac cgg gac gtg ctc aag gag atg aag gcg aag gcg tcc336 Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser 100105 110 aca gtt aag gcc aaa cta cta tca gta gaa gaa gcc tgt aag ctg acg384 Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr 115120 125 ccc cca cat tcg gcc aaa tcc aag ttt ggc tat ggg gca aag gac gtc432 Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val 130135 140 cgg aac cta tcc agc aag gcc gtt gac cac atc cgc tcc gtg tgg aag480 Arg Asn Leu Ser Ser Lys Ala Val Asp His Ile Arg Ser Val Trp Lys 145150 155 160 gac ttg ctg gaa gac act gag aca cca att gac acc acc atc atggcg 528 Asp Leu Leu Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala165 170 175 aaa aat gag gtt ttc tgc gtc caa cca gag aaa gga ggc cgc aagcca 576 Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro180 185 190 gct cgc ctt atc gta ttc cca gac ctg gga gtt cgt gta tgc gagaaa 624 Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys195 200 205 atg gcc ctc tac gac gtg gtc tcc acc ctt cct cag gcc gtg atgggc 672 Met Ala Leu Tyr Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly210 215 220 tcc tca tac gga ttc caa tac tct ccc aag cag cgg gtc gag ttcctg 720 Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Lys Gln Arg Val Glu Phe Leu225 230 235 240 gtg aat gcc tgg aaa tca aag aaa tgc cct atg ggc ttc tcatat gac 768 Val Asn Ala Trp Lys Ser Lys Lys Cys Pro Met Gly Phe Ser TyrAsp 245 250 255 acc cgc tgt ttt gac tca acg gtc act gag agc gac atc cgtgtt gag 816 Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg ValGlu 260 265 270 gag tca atc tac caa tgt tgt gac ttg gcc ccc gaa gcc agacag gct 864 Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg GlnAla 275 280 285 ata aag tcg ctc aca gag cgg ctc tat atc ggg ggt ccc ctgacc aat 912 Ile Lys Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu ThrAsn 290 295 300 tca aaa ggg cag aac tgc ggc tat cgc cgg tgc cgc gcg agcggc gtg 960 Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser GlyVal 305 310 315 320 ctg acg acc agc tgc ggt aat acc ctc aca tgt tac ttgaag gcc tct 1008 Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu LysAla Ser 325 330 335 gcg gcc tgt cga gct gcc aag ctc cag gac tgc acg atgctc gtg aac 1056 Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met LeuVal Asn 340 345 350 gga gac gac ctt gtc gtt atc tgc gag agc gcg gga acccag gag gat 1104 Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr GlnGlu Asp 355 360 365 gcg gcg aac cta cga gtc ttc acg gag gct atg act aggtac tct gcc 1152 Ala Ala Asn Leu Arg Val Phe Thr Glu Ala Met Thr Arg TyrSer Ala 370 375 380 ccc ccc ggg gac ctg ccc caa cca gaa tac gac ttg gagttg ata aca 1200 Pro Pro Gly Asp Leu Pro Gln Pro Glu Tyr Asp Leu Glu LeuIle Thr 385 390 395 400 tca tgc tcc tcc aat gtg tcg gtc gcg cac gat gcatcc ggc aaa agg 1248 Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Ala SerGly Lys Arg 405 410 415 gtg tac tac ctc acc cgt gac ccc acc acc ccc cttgcg cgg gct gcg 1296 Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu AlaArg Ala Ala 420 425 430 tgg gag aca gct aga cac act cca atc aac tcc tggcta ggc aat atc 1344 Trp Glu Thr Ala Arg His Thr Pro Ile Asn Ser Trp LeuGly Asn Ile 435 440 445 atc atg tat gcg ccc acc tta tgg gca agg atg gttctg atg act cat 1392 Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg Met Val LeuMet Thr His 450 455 460 ttc ttc tcc atc ctc cta gcc cag gag caa ctt gaaaaa gcc ctg gat 1440 Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln Leu Glu LysAla Leu Asp 465 470 475 480 tgt cag atc tac ggg gcc tgt tac tcc att gagcca ctt gac cta cct 1488 Cys Gln Ile Tyr Gly Ala Cys Tyr Ser Ile Glu ProLeu Asp Leu Pro 485 490 495 cag atc att gaa cga ctc cac ggt ctt agc gcattt tca ctc cac agt 1536 Gln Ile Ile Glu Arg Leu His Gly Leu Ser Ala PheSer Leu His Ser 500 505 510 tac tct cca ggt gaa atc aat agg gtg gct tcatgc ctc agg aaa ctt 1584 Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Ser CysLeu Arg Lys Leu 515 520 525 ggg gta cca ccc ttg cga gtc tgg aga cat cgggcc aga agt gtc cgc 1632 Gly Val Pro Pro Leu Arg Val Trp Arg His Arg AlaArg Ser Val Arg 530 535 540 gct aag ctg ctg tcc cag ggg ggg agg gct gccact tgt ggc aag tac 1680 Ala Lys Leu Leu Ser Gln Gly Gly Arg Ala Ala ThrCys Gly Lys Tyr 545 550 555 560 ctc ttc aac tgg gca gta agg acc aag cttaaa ctc act cca atc ccg 1728 Leu Phe Asn Trp Ala Val Arg Thr Lys Leu LysLeu Thr Pro Ile Pro 565 570 575 gct gcg tcc cgg ctg gac ttg tcc ggc tggttc gtt gct gga tac aac 1776 Ala Ala Ser Arg Leu Asp Leu Ser Gly Trp PheVal Ala Gly Tyr Asn 580 585 590 ggg gga gac ata tat cac agc ctg tct cgtgcc cga ccc cgt tgg ttt 1824 Gly Gly Asp Ile Tyr His Ser Leu Ser Arg AlaArg Pro Arg Trp Phe 595 600 605 atg ttg tgc cta ctc cta ctt tct gta ggggta ggc att tac ctg ctc 1872 Met Leu Cys Leu Leu Leu Leu Ser Val Gly ValGly Ile Tyr Leu Leu 610 615 620 ccc aac cga tga 1884 Pro Asn Arg 625 12627 PRT Artificial Sequence misc_feature HTaA5B polymerase 12 Met SerTyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr 1 5 10 15 ThrGlu Asn Leu Tyr Phe Gln Gly Ala Met Asp Pro Glu Phe Glu Asp 20 25 30 ValVal Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr 35 40 45 ProCys Ala Ala Glu Glu Ser Gln Leu Pro Ile Asn Ala Leu Ser Asn 50 55 60 SerLeu Val Arg His Arg Asn Met Val Tyr Ser Thr Thr Ser Arg Ser 65 70 75 80Ala Ala Leu Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu 85 90 95Asp Asp His Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser 100 105110 Thr Val Lys Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr 115120 125 Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val130 135 140 Arg Asn Leu Ser Ser Lys Ala Val Asp His Ile Arg Ser Val TrpLys 145 150 155 160 Asp Leu Leu Glu Asp Thr Glu Thr Pro Ile Asp Thr ThrIle Met Ala 165 170 175 Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys GlyGly Arg Lys Pro 180 185 190 Ala Arg Leu Ile Val Phe Pro Asp Leu Gly ValArg Val Cys Glu Lys 195 200 205 Met Ala Leu Tyr Asp Val Val Ser Thr LeuPro Gln Ala Val Met Gly 210 215 220 Ser Ser Tyr Gly Phe Gln Tyr Ser ProLys Gln Arg Val Glu Phe Leu 225 230 235 240 Val Asn Ala Trp Lys Ser LysLys Cys Pro Met Gly Phe Ser Tyr Asp 245 250 255 Thr Arg Cys Phe Asp SerThr Val Thr Glu Ser Asp Ile Arg Val Glu 260 265 270 Glu Ser Ile Tyr GlnCys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala 275 280 285 Ile Lys Ser LeuThr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn 290 295 300 Ser Lys GlyGln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val 305 310 315 320 LeuThr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser 325 330 335Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val Asn 340 345350 Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp 355360 365 Ala Ala Asn Leu Arg Val Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala370 375 380 Pro Pro Gly Asp Leu Pro Gln Pro Glu Tyr Asp Leu Glu Leu IleThr 385 390 395 400 Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Ala SerGly Lys Arg 405 410 415 Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro LeuAla Arg Ala Ala 420 425 430 Trp Glu Thr Ala Arg His Thr Pro Ile Asn SerTrp Leu Gly Asn Ile 435 440 445 Ile Met Tyr Ala Pro Thr Leu Trp Ala ArgMet Val Leu Met Thr His 450 455 460 Phe Phe Ser Ile Leu Leu Ala Gln GluGln Leu Glu Lys Ala Leu Asp 465 470 475 480 Cys Gln Ile Tyr Gly Ala CysTyr Ser Ile Glu Pro Leu Asp Leu Pro 485 490 495 Gln Ile Ile Glu Arg LeuHis Gly Leu Ser Ala Phe Ser Leu His Ser 500 505 510 Tyr Ser Pro Gly GluIle Asn Arg Val Ala Ser Cys Leu Arg Lys Leu 515 520 525 Gly Val Pro ProLeu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg 530 535 540 Ala Lys LeuLeu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr 545 550 555 560 LeuPhe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro 565 570 575Ala Ala Ser Arg Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Asn 580 585590 Gly Gly Asp Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe 595600 605 Met Leu Cys Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu610 615 620 Pro Asn Arg 625 13 45 DNA Artificial Sequence misc_featureoligonucleotide 13 ccaataggcc attttttttt tttttttttc tttccttctt tggtg 4514 42 DNA Artificial Sequence misc_feature oligonucleotide 14 ggaaagaaaaaaaaaaaaaa aaaatggcct attggcctgg ag 42 15 41 DNA Artificial Sequencemisc_feature oligonucleotide 15 cccctttttt tttttttctt tttttctttggtggctccat c 41 16 41 DNA Artificial Sequence misc_featureoligonucleotide 16 gccaccaaag aaaaaaagaa aaaaaaaaaa aggggatggc c 41 1745 DNA Artificial Sequence misc_feature oligonucleotide 17 ccaataggccattttttttt tttttttttc tttttttctt tggtg 45 18 45 DNA Artificial Sequencemisc_feature oligonucleotide 18 caccaaagaa aaaaagaaaa aaaaaaaaaaaaaatggcct attgg 45 19 350 DNA Artificial Sequence misc_feature template24-1 (m1) 19 gggagaccca agcttaaact cactccaatc ccggctgcgt cccggctggacttgtccggc 60 tggttcgttg ctggatacaa cgggggagac atatatcaca gcctgtctcgtgcccgaccc 120 cgttggttta tgttgtgcct actcctactt tctgtagggg taggcatttacctgctcccc 180 aaccgatgaa cggggggcta aacactccag gccaataggc catttttttttttttttttt 240 ctttccttct ttggtggctc catcttagcc ctagtcacgg ctagctgtgaaaggtccgtg 300 agccgcatga ctgcagagag tgctgatact ggcctctctg cagatcatgt350 20 350 DNA Artificial Sequence misc_feature template 24-1 (m2) 20gggagaccca agcttaaact cactccaatc ccggctgcgt cccggctgga cttgtccggc 60tggttcgttg ctggatacaa cgggggagac atatatcaca gcctgtctcg tgcccgaccc 120cgttggttta tgttgtgcct actcctactt tctgtagggg taggcattta cctgctcccc 180aaccgatgaa cggggggcta aacactccag gccaataggc catccccttt tttttttttt 240ctttttttct ttggtggctc catcttagcc ctagtcacgg ctagctgtga aaggtccgtg 300agccgcatga ctgcagagag tgctgatact ggcctctctg cagatcatgt 350 21 350 DNAArtificial Sequence misc_feature template 24-1 (dm) 21 gggagacccaagcttaaact cactccaatc ccggctgcgt cccggctgga cttgtccggc 60 tggttcgttgctggatacaa cgggggagac atatatcaca gcctgtctcg tgcccgaccc 120 cgttggtttatgttgtgcct actcctactt tctgtagggg taggcattta cctgctcccc 180 aaccgatgaacggggggcta aacactccag gccaataggc catttttttt tttttttttt 240 ctttttttctttggtggctc catcttagcc ctagtcacgg ctagctgtga aaggtccgtg 300 agccgcatgactgcagagag tgctgatact ggcctctctg cagatcatgt 350 22 25 RNA ArtificialSequence misc_feature RNA initiation site 22 uuuuuuuuuu uuucuuuccu ucuuu25 23 101 RNA Artificial Sequence misc_feature RNA initiation site 23uuuuuuuuuu uguuuuuuuu uuuuuuuuuu uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu 60uuuuuuuuuu uuccuuuucc uuuuuuuuuu auauuccuuc u 101 24 93 RNA ArtificialSequence misc_feature RNA initiation site 24 uuuuuuuuuu uuuugguuuuuuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60 uuuccuuuuu ccuuuuuuuuuuauauuccu ucu 93 25 98 RNA Artificial Sequence misc_feature RNAinitiation site 25 uuuuuuuuuu uuuguuuuuu uuuuuuuuuu uuuuuuuuuuuuuuuuuuuu uuuuuuuuuu 60 uuuuuuuuuu uuccuuuuuu uuuuuuuaua uuccuucu 98

What is claimed is:
 1. An isolated de novo initiation site for anRNA-dependent RNA polymerase (RdRp), said site consisting essentially ofa polypyrimidine tract and at least one cytidylate residue locatedtherein, or adjacent thereto.
 2. An isolated de novo initiation site foran RNA-dependent RNA polymerase, said site consisting essentially of apolypyrimidine tract comprising at least two or more adjacent cytidylateresidues located therein, or adjacent thereto.
 3. An isolated de novoinitiation site for an RNA-dependent RNA polymerase, said siteconsisting essentially of a polypyrimidine tract comprising a cluster ofcytidylate residues located therein, or adjacent thereto.
 4. The de novoinitiation site according to anyone of claims 1 to 3 , wherein said RdRpis a flavivirus polymerase.
 5. The de novo initiation site according toclaim 4 , wherein said polymerase is the Hepatitis C virus (HCV) NS5BRNA-dependent RNA polymerase, an analog, variant or derivative thereof.6. The RNA initiation site according to claim 1 , wherein saidpolypyrimidine tract essentially consists of greater than 90% ofuridylate residues.
 7. The RNA initiation site according to claim 1 ,comprising a sequence selected from the group consisting of:(P)_(n)(C)_(m); (C)_(m)(P)_(n); or (P)_(n)(C)_(m)(P)_(n), wherein P is apyrimidine or an analog thereof, each n is independently 2 to 200 and mis 1 to
 10. 8. The RNA initiation site according to claim 7 , whereinsaid P is polypyrimidine tract consisting essentially of greater than90% uridylate residues.
 9. The RNA molecule according to claim 1 ,comprising a CCC or CCCC sequence adjacent to a poly (U) tract.
 10. Anisolated de novo initiation site for an RNA-dependent RNA polymerase(RdRp), said site selected from the group consisting of: U₁₃CU₃CCUUCU₃(SEQ ID. NO. 21) U₁₁GU₂₉CU₃₀CCU₄CCU₁₀AUAUUCCUUCU (SEQ ID. NO. 22);U₁₄GGU₄₇CCU₅CCU₁₀AUAUUCCUUCU (SEQ ID. NO. 23); andU₁₃GU₅₈CCU₁₃AUAUUCCUUCU (SEQ ID. NO. 24).
 11. A RNA template forprimer-independent RNA synthesis comprising the isolated de novoinitiation site according to any one of claims 1 to 3 and 10 and afurther RNA portion suitable for providing a template for elongation ofsaid polymerase.
 12. A method of identifying a compound that inhibitsprimer-independent de novo RNA synthesis catalyzed by an RNA-dependentRNA polymerase (RdRp), comprising the steps of: a) contacting a RNAtemplate according to claim 11 , with said NS5B polymerase in theabsence of a primer and in the absence of said compound under conditionspermitting RNA synthesis, and determining the amount of RNA thus formed;b) contacting a RNA template as in a), with said RdRp in the absence ofa primer and in the presence of said compound under the same conditionsas in a), and determining the amount of RNA thus formed; and c)comparing the amount of RNA product formed in b) with that of a);wherein any reduction in the amount of RNA product formed in b) comparedwith that formed in a) indicates a compound that is an inhibitor ofprimer-independent de novo RNA synthesis catalyzed by said polymerase.13. The method according to claim 12 , wherein said polymerase is aflavivirus RdRp.
 14. The method according to claim 12 , wherein saidpolymerase is the Hepatitis C virus NS5B polymerase.
 15. The methodaccording to claim 12 , wherein said compound inhibits binding of saidNS5B polymerase to said initiation site of said template.
 16. The methodaccording to claim 12 , wherein said compound inhibits priming of saidNS5B polymerase once bound to said initiation site of said template. 17.The method according to claim 12 , wherein said compound inhibitselongation by said RNA polymerase along said further RNA portion of saidtemplate.
 18. A method of inhibiting primer- independent de novo RNAsynthesis catalyzed by an RNA-dependent RNA polymerase (RdRp) comprisingthe step of: contacting said polymerase with a polymerase-inhibitingeffective amount of a compound that inhibits primer-independent de novoRNA synthesis catalyzed by said polymerase, wherein said compound isidentified by the method according to claim 12 .
 19. The methodaccording to claim 18 , wherein said polymerase is a flavivirus RdRp.20. The method according to claim 18 , wherein said polymerase is theHepatitis C virus NS5B polymerase.
 21. The method according to claim 18, wherein said method provides inhibition of binding of said NS5Bpolymerase to said initiation site of said template.
 22. The methodaccording to claim 18 , wherein said method provides inhibition ofpriming of said NS5B polymerase once bound to said initiation site ofsaid template.
 23. The method according to claim 18 , wherein saidmethod provides inhibition of elongation by said RNA polymerase alongsaid further RNA portion of said template.
 24. A method of inhibitingthe replication of hepatitis C virus comprising the step of: a)contacting said hepatitis C virus with an antiviral effective amount ofa compound that inhibits primer-independent de novo RNA synthesiscatalyzed by HCV NS5B polymerase, wherein said compound is identified bythe method according to claim 12 .
 25. The method according to claim 24, wherein said method provides inhibition of binding of said NS5Bpolymerase to said initiation site of said template.
 26. The methodaccording to claim 24 , wherein said method provides inhibition ofpriming of said NS5B polymerase once bound to said initiation site ofsaid template.
 27. The method according to claim 24 , wherein saidmethod provides inhibition of elongation by said RNA polymerase alongsaid further RNA portion of said template.
 28. A method for producing aanti-HCV compound comprising the step of: identifying said compoundaccording to the method of claim 12 .
 29. A method of identifying acompound that inhibits primer-independent de novo RNA synthesiscatalyzed by a flavivirus RNA-dependent RNA polymerase, comprising: a)contacting a synthetic heteropolymeric RNA template comprising a clusterof cytidylate nucleotides or a mixed cluster of cytidylate and uridylatenucleotides with said polymerase in the absence of a primer and in theabsence of said compound under conditions permitting RNA synthesis, anddetermining the amount of RNA product thus formed; b) contacting an RNAtemplate as in a) with said polymerase in the absence of a primer and inthe presence of said compound under the same conditions as in a), anddetermining the amount of RNA product thus formed; and c) comparing theamount of RNA product formed in b) with that in a), wherein anyreduction in the amount of RNA product formed in b) compared with thatformed in a) indicates a compound that is an inhibitor ofprimer-independent de novo RNA synthesis catalyzed by said polymerase.30. The method according to claim 24 , wherein said polymerase is theHepatitis C virus NS5B RNA-dependent RNA polymerase, an analog, variantor derivative thereof.